Figure 5.
Figure 5. Induction of licensing markers in human monocytes by GM-CSF in vitro and ex vivo from tumor patients. (A) p-S6 and p-AKT expression detected by flow cytometry in fresh (day 1) and GM-CSF–cultured (day 7) human monocytes with or without IFN-γ stimulation. Background-subtracted MFI of p-S6 and p-AKT expression in fresh and GM-CSF–cultured human monocytes with or without IFN-γ stimulation (n = 3; mean ± SD). (B) IRF1 and IDO expression detected by flow cytometry in fresh (day 1) and cultured (day 7) human monocytes with or without IFN-γ stimulation. Background-subtracted MFI of IRF1 and IDO expression in fresh and cultured human monocytes with or without IFN-γ stimulation (n = 3; mean ± SD). (C) Effect of 10 ng/mL rapamycin over 7 days of culture on expression of p-S6, p-AKT, IRF1, and IDO in human monocytes (n = 3; mean ± SD). (D) Freshly isolated human CD14+ monocytes or monocytes cultured for 1 to 3 days in GM-CSF were added to phytohemagglutinin (PHA)-stimulated allogeneic human CD3+ T cells at a ratio 1:1 ratio for 4 days before determining CFSE dilution by FACS analysis and its quantification of T-cell suppression for human CD4+ and CD8+ T cells (human n = 6, statistics by ANOVA with multiple comparisons, *P < .05, ***P < .005). (E) Rapamycin (rapa)-treated human L-Mono were less effective than untreated L-Mono in suppressing PHA-stimulated proliferation of allogeneic human T cells in 1:1 direct cocultures (n = 3; mean ± SD). (F) Example dot plots of peripheral blood mononuclear cells from healthy individuals or tumor patients were stained for HLA-DR and CD14 to identify CD14+ HLA-DRlow M-MDSC (gate) by FACS analysis. (G) Cells gated as in panel F of healthy individuals or tumor patients were further costained for intracellular isotype controls, IDO, p-AKT, p-S6, or p-mTOR. FACS histograms represent examples of the indicated staining. The bar graph summarizes MFI values of the indicated markers with background-subtracted ΔMFI values from healthy (n = 4) or tumor patients (n = 5) and evaluated by unpaired Student t test. (H) Serum samples of (n = 51) tumor patients or (n = 9) healthy donors were analyzed for their GM-CSF (Mann-Whitney U test, mean ± SEM) or (I) for their M-CSF content from (n = 38) tumor patients or (n = 7) healthy donors (Mann-Whitney U test, mean ± SEM). Tumor patients analyzed in panels F and G were different from patients analyzed in panels H and I.

Induction of licensing markers in human monocytes by GM-CSF in vitro and ex vivo from tumor patients. (A) p-S6 and p-AKT expression detected by flow cytometry in fresh (day 1) and GM-CSF–cultured (day 7) human monocytes with or without IFN-γ stimulation. Background-subtracted MFI of p-S6 and p-AKT expression in fresh and GM-CSF–cultured human monocytes with or without IFN-γ stimulation (n = 3; mean ± SD). (B) IRF1 and IDO expression detected by flow cytometry in fresh (day 1) and cultured (day 7) human monocytes with or without IFN-γ stimulation. Background-subtracted MFI of IRF1 and IDO expression in fresh and cultured human monocytes with or without IFN-γ stimulation (n = 3; mean ± SD). (C) Effect of 10 ng/mL rapamycin over 7 days of culture on expression of p-S6, p-AKT, IRF1, and IDO in human monocytes (n = 3; mean ± SD). (D) Freshly isolated human CD14+ monocytes or monocytes cultured for 1 to 3 days in GM-CSF were added to phytohemagglutinin (PHA)-stimulated allogeneic human CD3+ T cells at a ratio 1:1 ratio for 4 days before determining CFSE dilution by FACS analysis and its quantification of T-cell suppression for human CD4+ and CD8+ T cells (human n = 6, statistics by ANOVA with multiple comparisons, *P < .05, ***P < .005). (E) Rapamycin (rapa)-treated human L-Mono were less effective than untreated L-Mono in suppressing PHA-stimulated proliferation of allogeneic human T cells in 1:1 direct cocultures (n = 3; mean ± SD). (F) Example dot plots of peripheral blood mononuclear cells from healthy individuals or tumor patients were stained for HLA-DR and CD14 to identify CD14+ HLA-DRlow M-MDSC (gate) by FACS analysis. (G) Cells gated as in panel F of healthy individuals or tumor patients were further costained for intracellular isotype controls, IDO, p-AKT, p-S6, or p-mTOR. FACS histograms represent examples of the indicated staining. The bar graph summarizes MFI values of the indicated markers with background-subtracted ΔMFI values from healthy (n = 4) or tumor patients (n = 5) and evaluated by unpaired Student t test. (H) Serum samples of (n = 51) tumor patients or (n = 9) healthy donors were analyzed for their GM-CSF (Mann-Whitney U test, mean ± SEM) or (I) for their M-CSF content from (n = 38) tumor patients or (n = 7) healthy donors (Mann-Whitney U test, mean ± SEM). Tumor patients analyzed in panels F and G were different from patients analyzed in panels H and I.

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